In the state of the art, different methods have been developed in microscopy to break the diffraction barrier. From WO 2006/127692 or DE 102006021317 A1, a method abbreviated to PALM (photoactivated localization microscopy) is known that uses a label substance for imaging a specimen, which label can be activated e.g. by means of optical radiation. Only in the activated state the label substance can emit particular fluorescent radiation. Non-activated label molecules do not emit any, or at least no noticeable fluorescent radiation, even after irradiation with excitation radiation. The activation radiation is therefore generally referred to as switchover signal and the molecules as photoswitches. In PALM, the switchover signal is applied in such a way that at least some activated labels are spaced apart from neighboring activated labels in such a way that—in terms of the optical resolution of the microscopy—these labels are separated or can be subsequently separated by image processing methods. It is said that a subset of the fluorescence emitters is isolated. An individual image is captured for the specimen prepared in this way. Therein, the center of the radiation distribution, which is caused by resolution limitation, of the isolated emitters is determined. The position of the molecules can be calculated therefrom with a higher degree of accuracy than is actually allowed by the optical resolution. This procedure is referred to as localization. The increased resolution is also referred to as “super resolution”. It requires that, in the specimen at least a subset of the activated labels can be distinguished, that is isolated within the given optical resolution. Then their location can be determined with greater accuracy; they can be localized.
The PALM method utilizes statistical effects to isolate individual label molecules. By setting the intensity of the switchover signal, it can be ensured that the probability of a label present in a given area of the specimen being activated is so low that there are sufficient sub-areas in which only labels can emit fluorescent radiation which can be distinguished within the optical resolution of the microscope. The sequence of isolation and localization is repeated several times, wherein each time a differently composed subset contributes to an individual image. The localization data from these individual images are finally compiled to form a high-resolving overall image.
The PALM method was refined, e.g., in DE 102008024568 A1 with respect to the activation of the labels to be detected. For this, fluorescent labels are used that have particular electronic states. They are activated by illumination radiation of high intensity in such a way that the great majority of the molecules are brought into an electronic, not fluorescent dark state. The remaining, then fluorescing molecules, can thus be isolated with respect to the optical resolution. The imaging and capture of the individual images is done as soon as a sufficient proportion of the molecules has been brought into the dark state, i.e. synchronized with the excitation. The exposure time for an individual image is based on the average time it takes to bring a label to the dark state by constant irradiation of excitation radiation. DE 102008024568 A1 requires a manipulation of the molecules in the specimen to the effect that the lifetime of the dark state is extended through the addition of chemical substances.
It is also noted that, in the meantime, modifications of PALM have been described in the technical literature which have received other abbreviations, such as for example STORM, dSTORM etc. Therefore, this description uses the term “localization microscopy” to cover all microscopy methods which achieve a spatial resolution beyond the optical resolution of the apparatus used by first isolating and then localizing fluorescent molecules.
As a rule, localization microscopy does not require a high spatial resolution for the illumination. A simple widefield illumination suffices in many cases. However, it requires many individual images of the specimen to be captured which in each case contain isolated molecules. In order to image the specimen completely, the total of all of the individual images must ensure that, as far as possible, all of the molecules were isolated in at least one individual image. For localization microscopy, the mentioned plurality of individual images must therefore routinely be captured, which involves a certain duration to generate an overall image.
Capturing time is also a problem for localization microscopy in that widefield detection, which is advantageous in and of itself, necessarily also requires a two-dimensional detector which must be sufficient sensitive to the detection of individual molecules. The sensitivity, usually indicated by the quantum efficiency, and the readout noise must be such that ideally individual, isolated molecules can be detected. This requirement sets for the cameras used in the state of the art in localization microscopy, a lower limit for the integration time in the capture of an individual image and thus an upper limit for the image refresh rate.
Time restraints are of particular concern when live-cell microscopy is to be carried out. In order to bring localization microscopy into a suitable time regime for this, it could be considered to take several molecules into evaluation for each diffraction-limited spot, therefore to broaden the term “isolated” to the effect that it is sufficient to isolate a group of molecules from neighboring molecule groups in terms of the resolution limit of the imaging, which limit is a diffraction-limited spot. Then, the density of the activated molecules could already be increased during the data capture and a given number of molecules could be detected more rapidly. However, in the case of current microscopy methods, this would be too much at the expense of localization accuracy.
DE 102008024568 A1 attempts to avoid the problem of the integration duration in that the lifetime of the dark state is increased by a chemical manipulation. A similar approach is taken by Heilemann et al., “Super-resolution imaging with small organic fluorophores.”, Angewandte Chemie International Edition, 48.37, (2009): 6903-6908. Here, the integration time can be set appropriately for the separation of individual molecule occurrences. However, the specimen then needs to be chemically manipulated.
So-called fluctuation analysis, which is also called SOFI, represents another route to high resolution. Here, individual fluorophores are caused to blink, and the blinking specimen is imaged in a widefield detection. The increase in resolution then takes place not through localization but through a correlation between the different blinking states. The approach of SOFI microscopy can be seen in analogy with recognition of a light-house in shipping. There, a particular light-house will differ from other light-houses in its blinking behavior. When, at one and the same location, blinking occurs which differs from the blinking of neighboring points, it is clear that there must be an emitter (light-house in shipping—fluorescent molecule in microscopy) at this location. SOFI microscopy requires that the correlated fluctuations must occur in a timescale that is accessible to the camera. This, finally, requires quite particular dyes or labels, e.g. so-called quantum dots, that blink slowly enough. These dyes are not very to not at all suitable for living specimens. In addition, here too there is the problem of a long measurement length since a certain minimum number of individual images are required for a relevant increase in resolution. As in DE 102008024568 A1, the integration period of the camera is also adapted to the duration of the bright state of the blinking emitters. Since SOFI microscopy evaluates correlations and operates without the steps of isolation and of localization, it does not count as localization microscopy. In addition, the possible increase in resolution is limited compared with localization microscopy. A further aspect in the case of live-cell microscopy is light damage; Waeldchen et al., Scientific Reports, 5:15348, DOI: 10.1038/srep15348, October 2015, show that irradiation with short wavelengths is many times more damaging than with longer-wave light. The switching wavelength used for PALM/dSTORM etc. is 405 nm, a further reason to dispense with photoswitches.